Regulable gene expression in Gram-positive bacteria

ABSTRACT

A system has been constructed which is suitable for tetracycline-inducible gene expression in Gram-positive bacteria such as  Staphylococcus aureus  and  Bacillus subtilis . The replicon/host gene expression system is tightly regulated, can be used in complex as well as minimal media, and can produce a high level of gene expression upon induction, with a variety of gene products. The gene expression system is suitable for production of products toxic to the host cells, and can be used, for example, for the analysis of gene expression and gene products in Gram-positive bacteria, and in a test of the effect of a peptide or polypeptide inhibitor of an  S. aureus  enzyme on the growth of  S. aureus  cells in culture or during infection of an animal.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/291,874 filed on Apr. 14, 1999, which is a continuation-in-partof U.S. patent application Ser. No. 09/227,687 filed on Jan. 8, 1999,which claims the benefit of U.S. provisional application Ser. No.60/070,965 filed on Jan. 9, 1998; U.S. provisional application Ser. No.60/076,638 filed on Mar. 3, 1998; U.S. provisional application Ser. No.60/081,753 filed on Apr. 14, 1998; U.S. provisional application Ser. No.60/085,844 filed on May 18, 1998; U.S. provisional application Ser. No.60/089,828 filed on Jun. 19, 1998; U.S. provisional application Ser. No.60/094,698 filed on Jul. 30, 1998; U.S. provisional application Ser. No.60/100,211 filed on Sep. 14, 1998; U.S. provisional application Ser. No.60/101,718 filed on Sep. 24, 1998; and U.S. provisional application Ser.No. 60/107,751 filed on Nov. 10, 1998. This application also claims thebenefit of U.S. provisional application Ser. No. 60/122,949 filed onMar. 5, 1999. The teachings of each of these referenced applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] A number of different high and low copy number vector systemsusing a diversity of regulable promoter systems have been successfullydeveloped to manipulate gene expression in Gram negative organisms suchas Escherichia coli. As a result, E. coli can be genetically manipulatedin a number of ways that have lead to a thorough understanding of themolecular basis for gene expression and to the elucidation of thefunction of many important proteins. As a further result, E. coli hasbeen used as a production organism for the high level expression of anumber of protein products, some of which are toxic. The majority ofthese vector systems developed in E. coli, however, do not functionproperly in Gram-positive microorganisms, likely due to physiologicaldifferences between Gram-positive and Gram-negative species (de Vos, W.M., et al., Curr. Opin. Biotechnol. 8:547-553, 1997; de Vos, W. M., andG. F. M. Simons, “Gene cloning and expression systems in lactococci,”pp. 52-105. In M. J. Gasson and W. M. de Vos (ed.) Genetics andBiotechnology of Lactic Acid Bacteria. Routledge, Chapman and Hall Inc.,New York, N.Y., 1994). The lack of vectors providing for the efficientlyregulated expression of genes in Gram-positive bacteria has beenresponsible, in part, for the lack of suitable Gram-positive systems forproduction of valuable gene products on an industrial scale.

[0003] The characterization of the biology of Gram-positive bacteria hasbeen hampered by the lack of cloning and expression vector systems thatare stably maintained, tightly regulated and inducible, analogous tothose developed in E. coli. As a result, the study of importantGram-positive pathogens, that can cause a variety of different illnessesincluding life threatening ones, has been severely limited, impeding thediscovery of novel, life saving therapies to treat infectious diseases.

[0004] In the last decade, the rapid rise in severe and fatal infectionscaused by drug resistant microbial pathogens has presented a significantthreat to public health worldwide. One of the pathogens of immediateconcern is the Gram-positive organism Staphylococcus aureus. There areover nine million cases of S. aureus infections a year. S. aureusinfections are one of the most prevalent types of hospital acquiredinfections requiring treatment. Methicillin resistant S. aureus nowrepresents a significant proportion of all Staphylococcus aureusinfections in hospitals. Although these infections can be treated withthe antibiotic vancomycin, it has been documented that S. aureus strainscan become resistant to this last line antibiotic. There is genuineconcern in the medical community that new antibiotics to treat S. aureusinfections must be discovered in order to prevent a return to thepreantibiotic era where death by bacterial infection was common.

[0005] A necessary first step towards the identification of noveltherapeutics to treat Staphylococcus aureus and other Gram-positivebacterial infections is an ability to clone and regulate the expressionof genes in the organism in order to understand its biology. To do this,replicons—replicating units of DNA or RNA, such as plasmids—should beconstructed to carry the genes and ensure the desired level of geneexpression when the replicons are in the bacterial cells. A system usingspecially constructed bacterial strains is needed to control andevaluate expression in vitro as well as in vivo during a bacterialinfection in an animal model system so that the biology of theinfectious disease process can be investigated.

[0006] To provide broad practical applications of a Gram-positiveinducible gene expression system, it is desirable for the system to havethe following features: (1) The system is under tight expression controlthat avoids or minimizes leaky expression of a cloned gene. Leakyexpression can often result in cell toxicity and so must be avoided. (2)The system responds with high levels of expression upon induction. (3)The inducibility of the system is independent of growth media so that avariety of environmental conditions can be evaluated. (4) Administeringinducer to the gene expression system, by itself (that is, withoutproduction in the cells of the regulated gene product) does not causesignificant change in the phenotype of the bacteria (e.g., inhibit thegrowth of the bacteria). (5) The system functions with features (1),(2), (3) and (4) not only when the bacteria are grown in culture, butalso in an animal infection model. To date, there has been no acceptableinducible expression system that covers the above listed criteria. Inaddition, if improved cloning and expression systems were available awealth of opportunities could be realized for the efficient and economicutilization of microorganisms for the industrial production ofmacromolecules.

SUMMARY OF THE INVENTION

[0007] The invention encompasses a replicon which can be used for thehigh-level, inducible production of a gene product in Gram-positivebacteria, including, but not limited to staphylococci (for instance, S.aureus and S. carnosus), and Gram-positive bacilli (for instance,Bacillus subtilis). The invention encompasses a replicon comprising apromoter/operator region that causes gene expression under its controlto be tightly repressed in the absence of tetracycline or an analog oftetracycline. However, in the presence of tetracycline or an analogthereof, the promoter/operator region causes a high level of geneexpression of a gene situated downstream of the promoter. That is, thepromoter/operator can produce a high level of transcription, but istightly regulated (non-leaky).

[0008] Built into the replicon is a linker site downstream of thepromoter/operator region so that insertion of a segment of nucleic acidencoding a gene product (wherein the segment comprises an open readingframe) at the linker site can put the transcription of such an openreading frame under the control of the promoter/operator region.Optionally, a linker site can be within or adjacent to a segment ofnucleic acid encoding a carrier polypeptide, such that insertion, inframe, of a nucleic acid segment encoding a second polypeptide (whichcan be as short as a few amino acid residues, and usually termed a“peptide,” but here, included in the term “polypeptide”) results in aderivative replicon, which when introduced into an appropriate hostspecies of bacteria, can inducibly produce a fusion polypeptide (also,“fusion protein”) of the carrier and second polypeptides.

[0009] A particular promoter/operator region, called P_(JT)/TetO herein,and which has been described herein as a part of pC³875, has been foundto be particularly well suited to the tightly controlledtetracycline-regulated expression of genes inserted downstream from thepromoter/operator. (Herein, “tetracycline-inducible” or“tetracycline-regulated” describes genetic elements responsive totetracycline as well as to analogs thereof that act similarly totetracycline.)

[0010] Also an aspect of the invention is a strain of Gram-positivebacteria comprising one or more genes encoding tetracycline resistance[tet(M), tet (0), etc.] and tetR (tet repressor) genes. Construction ofsuch a strain of S. aureus is described herein.

[0011] A further aspect of the invention is a system for inducibleexpression of a gene, comprising Gram-positive bacteria bearing areplicon, wherein the replicon comprises a tetracycline-induciblepromoter/operator region for the tightly regulated control of geneexpression, for example, the promoter/operator P_(JT)/TetO (for tetoperator, originally described in transposon Tn10, see Wissmann, A. etal., J. Mol. Biol. 101:397-406, 1988; also Geissendorfer, M. and W.Hillen, Appl. Microbiol. Biotechnol. 33:657-663, 1990), and furthercomprising an open reading frame downstream of said promoter/operatorregion, wherein the open reading frame can be a coding sequence for apolypeptide, which can be a fusion polypeptide, for example. TheGram-positive bacteria can be S. aureus, for instance.

[0012] The replicon of the gene expression system can be constructed tohave, instead of an open reading frame immediately downstream of thepromoter/operator region, a linker site for the insertion of a segmentof nucleic acid comprising a coding region for a gene of interest. Thereplicon of the gene expression system can also be constructed to have alinker site downstream of the promoter/operator region, wherein thelinker is within or adjacent to a coding region for a carrierpolypeptide, so that insertion of a second coding sequence results intetracycline-inducible production of a “carrier-second” fusionpolypeptide in cells bearing the replicon.

[0013] The system of gene expression described herein is especially wellsuited to the production of and/or analysis of the effects of geneproducts that may be toxic to the host cells. The system has furtheradvantages in being suitable for use in an animal testing method. Inthis method, animals are infected with a strain of engineered bacteria,such as S. aureus (herein, the strain of engineered bacteria is an“inducible system for expression of a gene”) where the inducible geneencodes a “carrier-peptide” fusion protein. The peptide portion of thefusion protein encoded by the bacterial replicon is a candidate forcausing a phenotypic effect on the host bacterial cells, typically, byenhancement or inhibition of the function of a host cell component, suchas the activity of a host bacterial cell enzyme, resulting in a slowingor cessation of growth of the host bacterial cells. Tetracycline orinducing derivatives or analogs thereof can be safely given to theinfected animals, to induce the controlled production of the“carrier-peptide” fusion protein. If the tetracycline induction of geneexpression of the “carrier-peptide” fusion protein produces a slowing orcessation of growth of bacterial cells infecting the test animals(resulting in animal survival), then the peptide portion of the fusionprotein is proven effective as having antibiotic action. It can beconcluded then, also, that the peptide portion is affecting a targetcell component that is essential to growth of the bacterial cells.Further measures can be taken to find more physiologically stablestructural analogs of the peptide or to otherwise develop antibioticsmodeled on the structure of the peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1B is a diagram of the steps taken to construct plasmidpCL84tt, which was then used in the construction of S. aureus strainCYL316. Restriction sites on the plasmids are indicated as follows:E=EcoRI; H=HindIII; S=SalI. P indicates promoter.

[0015]FIG. 2 is a diagram of the steps taken to construct plasmidspC³874 and pC³875.

[0016]FIG. 3A is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316t in the presence (open diamonds) or absence (filleddiamonds) of 200 ng/ml of anhydrotetracycline.

[0017]FIG. 3B is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316t/pWH353(ΔtetR) in the presence (open diamonds) orabsence (filled diamonds) of 200 ng/ml of anhydrotetracycline.

[0018]FIG. 3C is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316t/pWH354(ΔtetR) in the presence (open diamonds) orabsence (filled diamonds) of 200 ng/ml of anhydrotetracycline.

[0019]FIG. 3D is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316t/pWH354-2G(ΔtetR) in the presence (open diamonds)or absence (filled diamonds) of 200 ng/ml of anhydrotetracycline.

[0020]FIG. 3E is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316t/pC³859 in the presence (open diamonds) or absence(filled diamonds) of 200 ng/ml of anhydrotetracycline.

[0021]FIG. 4 is a scan of an SDS-polyacrylamide gel, stained withCoomassie blue, which was loaded with the glutathione-Sepharose-purifiedproteins produced under induction conditions (“I” lanes) or uninducedconditions (“U” lanes) in CYL316t/pC³878, CYL316t/pC³876, CYL316t/pC³874and CYL316t/pC³875. Glutathione S-transferase is the prominent band.

[0022]FIG. 5A is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316tt bearing pC³875 upon induction (filled diamonds)or no induction (open diamonds) of gene expression under control of thetetracycline promoter/operator.

[0023]FIG. 5B is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316tt bearing pC³882 upon induction (filled diamonds)or no induction (open diamonds) of gene expression under control of thetetracycline promoter/operator.

[0024]FIG. 5C is a graph showing the growth, as measured by OD₆₀₀, of S.aureus strain CYL316tt bearing pC³883 upon induction (filled diamonds)or no induction (open diamonds) of gene expression under control of thetetracycline promoter/operator.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention includes replicons suited to use for the expressionof a desired gene and the production of one or more gene products,wherein the gene product can be mRNA, tRNA, rRNA, but is preferably apolypeptide (e.g., a peptide or a protein, such as an enzyme) includinga fusion polypeptide, in bacteria. Replicons can include, for example,plasmids, phagemids, cosmids, genetically engineered bacteriophages orviruses of appropriate host range. A replicon can be single-stranded ordouble-stranded DNA or RNA or modified variants thereof, which can, forsome purposes, include modified or synthesized bases, and bases notfound in nucleic acids isolated from sources found in nature. A repliconcan be a discrete unit of nucleic acid that replicates independently ofthe chromosome of its host bacterial cell, (“replicon” herein excludesthe chromosome of the bacterial cell) and can be present as a singlecopy or as multiple copies. In each case, the elements necessary forreplication of the replicon nucleic acid are found in the nucleic acidof the replicon or can be found in the host bacterial cells containingthe replicon, so that the replicon (“replicon” encompassing the plural,as a great number are usually present in cultures of cells or as theisolated nucleic acid) can be produced within the bacterial cells fromone generation to the next, or can be produced and recovered in a formthat can be introduced into bacterial cells of a culture different fromthat in which they reproduced, to initiate further rounds ofreplication.

[0026] Gram-positive bacteria are those which can be identified by thewell-known Gram staining method, and include, for example,Staphylococcus aureus, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcussalivarius, Streptococcus mitior, Streptococcus milleri, Streptococcussanguis, Streptococcus mutans, Corynebacterium diphtheriae,Corynebacterium hemolyticum, Enterococcus faecalis, Enterococcusfaecium, Enterococcus avium, Enterococcus casseliflavus, Enterococcusdurans, Enterococcus gallinarum, Enterococcus molodoratus, Enterococcusaffinosus, Enterococcus pseudoavium, Enterococcus solitarius,Enterococcus mundtii, Enterococcus hirae, Listeria monocytogenes,Bacillus anthracis, Clostridium perfringens, and Clostridium difficile.

[0027] The replicon of the invention comprises several elements whichare encoded in the order of nucleotides of the nucleic acid of thereplicon. These elements can be sites necessary for replication of thereplicon, such as an origin of replication, having one or more sites forrecognition by a DNA polymerase. Other sites can include apromoter/operator region, which can have one or more sites to which arepressor can bind (operator) to regulate transcription initiating atthe promoter, and one or more sites for recognition of RNA polymerase(promoter). Downstream from the promoter (that is, in the direction oftravel of RNA polymerase from the promoter during transcription—5′ to 3′with respect to the sense strand) can lie a linker site, which can beone or more restriction sites (cleavable by use of one or morerestriction enzymes under conditions appropriate for restriction enzymeactivity). A linker site can be positioned such that it lies outside ofany desired coding sequence, in which case it can be used for theinsertion of a segment of nucleic acid having a sequence encoding a geneproduct. In an alternative arrangement, for producing a fusionpolypeptide, a linker site can be positioned such that it lies within oradjacent to a first sequence encoding a first polypeptide (which issometimes called a “carrier” protein or polypeptide), such thatinsertion into the linker site of a segment of nucleic acid having asecond sequence encoding a second polypeptide results in a gene whichencodes, under the regulation of the promoter/operator region, a fusionpolypeptide having an amino acid sequence encoded, in part, by the firstnucleic acid sequence, and in part, by the second nucleic acid sequence.

[0028] Some embodiments of the replicon are nucleic acid replicons thatreplicate in host cells of one or more species of Gram-positivebacteria, each replicon comprising an inducible gene which does notencode chloramphenicol acetyltransferase or tet repressor, whereinexpression of said gene is non-leaky, and wherein expression of saidgene is high level in the host cells under inducing conditions.

[0029] A further embodiment is a nucleic acid replicon that replicatesin one or more species of bacteria, said replicon comprising atetracycline-inducible promoter/operator region, wherein the promoter ofsaid promoter/operator region does not control production of tetrepressor or chloramphenicol acetyltransferase, and further comprising alinker site downstream of the promoter/operator region such thatinsertion, in frame, of a nucleic acid segment encoding a gene productresults in a derivative replicon in which production of the gene productin the bacteria is under tetracycline-inducible control by thetetracycline-inducible promoter/operator region in said replicon.

[0030] The term “polypeptide” is not intended to be limiting as to thenumber of amino acid residues. Peptides as short as three amino acidresidues are included within the term “polypeptide” as used herein.Polypeptides can have amino acid sequences that are the same as, orsimilar to, those of proteins and polypeptides found in natural sources,or they can have amino acid sequences that are the product of theinvention of humans. Polypeptides produced in cells can also havepost-translational modifications such as phosphorylation or removal of aportion by enzymatic cleavage.

[0031] It is known that many antibiotics exert their physiologicaleffect by binding to, and inhibiting the function of, a component of acell which is the target of treatment. For example, the target cellcomponent can be an enzyme, and the inhibitor can bind to the activesite, or near to, the active site so as to inhibit the activity of theenzyme. The inhibitor can also, for example, bind to a site on a proteinnecessary for interaction with another protein or a cofactor which isnecessary for normal function of the protein. The result of binding ofan inhibitor to its target site is a phenotypic effect on the cell(“cell” includes cells of a cell culture or cell strain), which asdesirable for an antibiotic, can be slowing or cessation of cell growth.In the case of an inhibitor which binds to and inhibits the function ofa target cell component of a species of pathogenic bacteria, without oronly minimally binding to and inhibiting the function of a cellcomponent of a mammal which can be infected by the pathogenic bacteria,the inhibitor is displaying a function as an antibiotic, and can be thebasis for further study (e.g., rational drug design based on thestructure of the inhibitor, or screening of libraries of compounds todiscover those that may have similar binding to the target site of theinhibitor).

[0032] A polypeptide to be encoded under the (transcriptional) controlof the promoter/operator region of the replicon, whether the polypeptideis a fusion polypeptide or is a nonfusion polypeptide, can be, forexample, a candidate molecule to be tested for inhibition of enzymeactivity of a host bacterial enzyme, upon induction of gene expressionand production of the polypeptide. The inducible expression controlledby the regulated promoter/operator region makes a replicon that issuitable for the expression of polypeptides that are toxic to the hostcells when made in sufficiently large quantities. Therefore, thereplicon of the invention is well suited to a test, in bacteria, ofpolypeptides that, as explained above, are candidates for havingantibiotic activity.

[0033] The first polypeptide of a fusion polypeptide can be, forexample, glutathione S-transferase (GST) of Schistosoma japonicum. GSTcoding sequences have been described and are available as part of genefusion vectors available from Pharmacia (Uppsala, Sweden). The firstpolypeptide of a fusion polypeptide can be, for example, greenfluorescence protein (GFP), maltose binding protein (MBP), protein A, orβ-galactosidase, cellulose binding domain (Novagen), chitin bindingdomain (New England Biolabs), a polypeptide including a His-Tag affinityligand (pET series plasmids, Novagen), Flag-Tag, or a sufficiently longportion of a polypeptide such that affinity binding characteristics ofthe carrier portion can be used to advantage in the purification of afusion protein comprising such portion of a carrier polypeptide.

[0034] The replicon can further comprise other components within itsnucleic acid, including, for example, a selectable marker gene. Forexample, a gene encoding a protein that confers upon the host bacterialcells resistance to an antibiotic, can be used as a device to ensuremaintenance of the replicon in the host cells over many generations ofgrowth of the host bacterial cells.

[0035] Also an embodiment of the invention is a system for theexpression of a gene, resulting in the production of a gene product,comprising host bacterial cells which comprise a replicon as describedabove. The system can carry, in addition to those genetic componentsincorporated into the replicon, genetic components, present in one ormore copies, incorporated into the chromosome of the host bacterialcells or carried on another replicon. The location of the geneticcomponents is not critical, except that the placement of thetetracycline promoter/operator region and the coding region to be underits control be such that production of the gene product is controlled bytetracycline induction of the default repressed condition. The distancebetween the promoter/operator region and the coding region or openreading frame can vary among effectively controlled systems, and can bedetermined by one of ordinary skill in the art. In the case of a systemcarrying on the replicon a promoter/operator region controllingtranscription by induction with tetracycline or a suitable analog oftetracycline, such as anhydrotetracycline, the system can haveincorporated into it a gene encoding a product which confers atetracycline resistant phenotype on the host bacterial cells, such asthe tet(M) tetracycline resistance gene (Burdett, V., J. Biol. Chem.266:2872-2877, 1991), or the tet(O) tetracycline resistance gene(Manavathu, E. K. et al., Gene (Amst.) 62:17-26, 1988). Such a gene canbe carried on the replicon, on the bacterial chromosome, or on someother extrachromosomal element.

[0036] The tet(M) tetracycline resistance gene was initially identifiedin streptococci (Burdett, V. et al., Microbiology 1982 (Schlessinger,D., ed.) pp. 155-158, American Society for Microbiology, Washington,D.C.), but has also been identified in staphylococci (Bismuth, R. etal., Antimicrob. Agents Chemother. 34:1611-1614, 1990), Clostridiadifficile (Hächler, H. et al., Antimicrob. Agents Chemother.31:1033-1038, 1987), Neisseria gonorrhoeae (Morse, S. A. et al.,Antimicrob. Agents Chemother. 30:664-670, 1986), and mycoplasmas(Roberts, M. C. et al, Antimicrob. Agents Chemother. 28:141-143, 1985);it has been cloned and expressed in Bacillus subtilis (Clewell, D. G.and Gawron-Burke, C., Annu. Rev. Microbiol. 40:635-659, 1986; Courvalin,P. and Carlier, C., Mol. Gen. Genet. 205:291-297, 1987) and Escherichiacoli (Burdett, V. et al., J. Bacteriol 149:995-1004, 1982). Other genesare known to produce a tetracycline resistant phenotype and can be usedin the gene expression system of the invention; a tet(O) gene has beencharacterized, in addition to tet(M), in Streptococcus faecalis(Burdett, V., J. Bacteriol. 165:564-569, 1986).

[0037] In addition to a gene conferring tetracycline resistance on thehost bacterial cells of the gene expression system, the system can alsocarry one or more genes which encode a repressor of thetetracycline-inducible promoter/operator (“tet repressor” or“tetracycline repressor”). Such repressor genes can be induciblyexpressed (for example, by a tet promoter/operator), but are preferablyconstitutively expressed.

[0038] The invention also provides a system for the production of a geneproduct that is tightly regulated or tightly controlled—that is, thesystem does not suffer from “leaky” gene expression. In this way, a geneproduct that causes some degree of toxicity to the cells, can beproduced in the cells of the system only when it is desired, and thegenetic content of the cells can be stably maintained from onegeneration to the next without the danger of selection against thosecells expressing a product which can inhibit growth and replication ofthe cells.

[0039] Preferably, the system responds to the induced condition with ahigh level of production of the gene product, relative to the uninducedcondition. The level of production of the gene product can be assayedby, for example, assays of enzymatic activity (e.g., aminoacylationactivity by an aminoacyl-tRNA synthetase), by quantitation of purifiedgene product (for instance, by determinations of protein concentration,quantitation of protein labeled with a radioactive isotope or afluorescent tag). In a preferred embodiment, the system responds toinduction of gene expression with a high level of gene expression, byproducing a high level of gene product (e.g., per unit of cells or celllysate, total cell protein, or total cell RNA, etc., as appropriate)which is at least about 10 times the uninduced level; in a morepreferred embodiment, at least about 100 times the uninduced level; in astill more preferred embodiment, at least about 1,000 times theuninduced level; and most preferably, at least about 10,000 times theuninduced level.

[0040] The invention also provides a system which can accommodate theproduction of a wide variety of gene products in Gram-positive bacteria,which has not been demonstrated, for example, with a system employingthe plasmids of Hillen et al. (DE 3934454 Al; Geissendörfer, M. and W.Hillen, Appl. Microbiol. Biotechnol. 33:657-663, 1990). A desirableproperty of the system of the invention is that administering inducer tothe gene expression system (even before, or in the absence of thebiosynthesis of the controlled gene product), does not cause asignificant change in the phenotype of the cells—that is, a change thatis an effect detrimental to growth or some effect that can mask theeffect on the phenotype of the induced gene product.

[0041] The invention further provides a system in which expression of agene of interest can be efficiently induced in rich, complete media [forexample, Luria-Bertani broth (LB), NZ-amine, Voges-Proskauer broth,nutrient broth, trypticase soy broth, Robertson's cooked meat medium;see, for instance, Bergey's Manual of Systematic Bacteriology, (P. H. A.Sneath et al., eds.), Williams and Wilkins, Baltimore, Md., 1986], aswell as in minimal media (essential salts, including a nitrogen source,and a sugar to supply a carbon source). Other systems for the regulatedexpression of a gene and production of a polypeptide have been describedin Gram-positive bacteria. For example, a pBla/BlaR system in S. aureushas been described, although this system has suffered from instabilityof plasmids. A system in B. subtilis has been described which employsthe E. coli lac operon (Yansura, D. G., and D. J. Henner, Proc. Natl.Acad. Sci. USA 81:439-443, 1984). A system for regulated gene expressionwhich employs an inducible xyl operon has been tried in staphylococci(Sizemore, C. et al., FEMS Microbiology Letters 107:303-306, 1993). Asystem for the nisin-inducible expression of a gene regulated by thelactococcal nisA promoter has been described for Streptococcus pyogenesand for Bacillus subtilis (Eichenbaum, Z. et al., Applied andEnvironmental Microbiology 64:2763-2769, 1998).

[0042] The invention also provides a gene expression system which can beused for the testing of the phenotypic effect of a gene product upon thehost bacterial cells in which its biosynthesis occurs, upon regulatedproduction of the gene product. The phenotypic effect of the geneproduct may be, for example, the enhancement or inhibition of activityof an enzyme, which, in some cases, can cause an inhibition of growth ofthe host bacterial cells.

[0043] The testing of phenotypic effect can be done in culture, usinginducer to turn on gene expression and thereby, production of the geneproduct. The phenotypic effect can be observed by various assays (e.g.,monitoring an enzymatic activity, growth rate, level of a metabolite orintermediate in a biosynthetic pathway) on the induced culture ofbacterial cells, comparing the results with those of an uninducedculture. For an optimal test of phenotypic effect, the basal (uninduced,or repressed) level of gene expression should be minimal, so that geneexpression is not “leaky.” That is, the basal level of the gene productof the regulated gene of a non-leaky system is such that the phenotypiceffect expected in the induced cells is not observed, or not observed toa significant extent (e.g., a statistically significant extent) overcells not containing the gene. It is preferable that the basal level ofthe gene product of the regulated gene of the system is undetectable asa band on an SDS-polyacrylamide gel stained with Coomassie blue, as itwas for an expression system containing pC³875 (see Example 3 and FIG.4). In one embodiment, the basal level of the encoded gene product ofthe gene expression system is less than about 100 μg/ml of initial wetcell weight; in preferred embodiment, less than about 10 μg/ml; in amore preferred embodiment, less than about 100 ng/ml, and in an evenmore preferred embodiment, less than about 10 ng/ml.

[0044] The testing of phenotypic effect can be done in an animal model,as well as in Gram-positive bacterial cells grown in culture. See U.S.patent application Ser. No. 09/227,687, the teachings of which areincorporated by reference herein. For an engineered strain ofGram-positive bacterial cells to be used in the gene expression systemof the invention, an object of the test can be to see whether productionof the gene product in the engineered strain inhibits growth of thesecells after the introduction of the cells into one or more animals.Suitable animals for such an experiment are, for example, mammals suchas mice, rats, rabbits, guinea pigs, dogs, pigs, and the like. Smallmammals are preferred for reasons of convenience. The engineeredGram-positive bacterial cells of the inducible gene expression systemare introduced into one or more animals (“test” animals) and into one ormore animals in a separate group (“control” animals) by a routeappropriate to cause symptoms of systemic or local growth of theengineered cells (infection). The route of introduction may be, forexample, by oral feeding, by inhalation, by subcutaneous, intramuscular,intravenous, or intraperitoneal injection as appropriate to the desiredresult (e.g., location and severity of infection).

[0045] After the Gram-positive bacterial cells of the inducible geneexpression system of the invention have been introduced into the testand control animals, expression of the gene encoding the gene product ofinterest is regulated to allow production of the gene product in theengineered pathogen cells. This can be achieved, for instance, byadministering to the test animals a treatment appropriate to theregulation system built into the cells, (e.g., administeringtetracycline or an appropriate analog such as anhydrotetracycline) tocause the gene encoding the gene product of interest to be expressed.The same treatment is not administered to the infected control animals,but the conditions under which they are maintained are otherwiseidentical to those of the test animals.

[0046] After such treatment, the test and control animals can bemonitored for a phenotypic effect in the introduced cells. The animalscan be monitored for signs of infection with the engineeredGram-positive bacterial cells of the tetracycline-inducible geneexpression system of the invention (as the simplest endpoint, death ofthe animal, but also e.g., lethargy, lack of grooming behavior, hunchedposture, not eating, diarrhea or other discharges; bacterial titer insamples of blood or other cultured fluids or tissues). If the testanimals are observed to exhibit less growth of the introduced cells thanthe control animals, then it can be concluded that the expression invivo of the gene product of the regulated gene is the cause of therelative reduction in growth of the introduced cells in the testanimals, and that the gene product is interfering with a target cellcomponent whose function is necessary to the normal growth of theintroduced cells.

[0047] “In culture” and “in animal” tests for the effect of a geneproduct expressed upon induction of the gene expression system asdescribed above can be done in succession, or concurrently. Following ananimal test as described above, further steps can be taken involving invitro assays to identify one or more compounds that have binding andactivating or inhibitory properties that are similar to those of thegene product which has been found to have a phenotypic effect whenproduced intracellularly, such as inhibition of growth. That is,compounds that compete for binding to a target cell component with thegene product tested would then be structural analogs of that geneproduct and could be, for example, candidates for antibiotic activity.Assays to identify such compounds can take advantage of known methods toidentify competing molecules in a binding assay.

[0048] Thus, the test in an animal model as described above is a methodthat can be used to confirm that a gene product, inducibly produced inthe Gram-positive bacteria, can function as an antibiotic underconditions of an infection. The system of inducible gene expressiondescribed herein (that is, the Gram-positive bacteria bearing repliconsas described herein) is especially well-suited to use in the test of theeffect of the inducible gene product, in an “in animal” test (infection)as well as in an “in culture” test. The system has the followingadvantages for an “in animal” test.

[0049] (1) The inducible gene expression system does not requirespecially-formulated media (for instance, a minimal salts medium or aspecial change of carbon source for induction). The inducible systemworks well in the rich medium supplied by animal biological fluids, aswell as in defined medium.

[0050] (2) The induction method alone has no measurable effect on mice.Nor does adding inducer (or an analog thereof that has the same effectas the inducer) to the bacteria of the gene expression system,alone—that is, in the absence of production of the induced geneproduct—cause any measurable phenotypic change in the bacteria.

[0051] (3) The inducible gene expression system as described herein isa) tightly controlled, and b) inducible to a high level of intracellulargene product, making the results of an “in animal” test clearlyinterpretable. Complete inhibition of the Gram-positive bacterial celltarget is possible for an effective inhibitor of an essential targetcell component. This can result in sparing of the infected test animalsin which gene expression of the regulated gene is induced, compared tono effect in the control animals (course of infection is not altered,resulting in death).

[0052] One of ordinary skill in the art can adapt the replicon describedherein to make use of the promoter/operator region (for instance, thepromoter/operator region of pC³875, designated P_(JT)/TetO) andoptionally, a coding region for a fusion polypeptide, by inserting itinto replicons having different origins of replication, and therefore,different host ranges. The P_(JT)/TetO-GST region of pC³875 can beexcised from pC³875 using EcoRI digestion or digestion with BstEII andSpeI, for insertion into sites of different replicons. See map of pC³875in FIG. 2.

[0053] A large number of plasmids capable of replicating in S. aureushave been identified, such as pC194, pT181, pSN2, and pE194 (Novick, R.P., Methods in Enzymology 204:587-637, 1991). Many of these plasmids arealso capable of replicating in other Gram-positive species (e.g.,Staphylococcus carnosus, Bacillus subtilis, and subspecies ofLactococcus). Additional examples of plasmids to be modified byinsertion of P_(JT)/TetO or P_(JT)/TetO together with an open readingframe of interest, include pAM401 (ATCC No. 37429; an E. faecalis/E.coli shuttle vector), and Streptococcus pneumoniae plasmids pLSE4 (ATCCNo. 37819), pLS21 (ATCC No. 67492), and pLS101 (ATCC No. 39938).

[0054] One of ordinary skill in the art recognizes that certainfunctional regions and nucleotide sequence features are common topromoters and operator regions of bacteria and that some of thesenucleotide sequence features are more particular to one species or groupof species. See, for example, Lewin, pages 302-305 In Genes, OxfordUniversity Press, 1997. Thus, it is possible to produce variants ofP_(JT)/TetO, which differ from in nucleotide sequence from that ofP_(JT)/TetO, but which retain the functional characteristics of as acontrol region. Variants include those promoter/operator regions thatdiffer from P_(JT)/TetO by the substitution, deletion, or insertion ofone or more nucleotides, especially within non-consensus sequences. Suchvariants of P_(JT)/TetO are also included in the invention.

EXAMPLES Example 1 Construction of Staphylococcus aureus Strains ThatConstitutively Express the Tet Repressor, TetR Construction ofpCL84(ΔTc, ΔH3)

[0055] pCL84 is a S. aureus chromosome integration plasmid (Lee, C. Y.et al., Gene 103:101-105, 1991) and was used for integration of the tetrepressor gene into the S. aureus chromosome. pCL84 carries atetracycline resistance gene (tet(K)) that is derived from plasmid pT181(Guay, G. G. et al., Plasmid 30:163-166, 1993). Since this tetracyclineresistance gene encodes a protein that acts as a tetracycline effluxpump, which is not desirable for a tetracycline inducible expressionsystem, the following steps were taken to eliminate this gene frompCL84. Plasmid construction steps are diagramed in FIGS. 1A-1B. pCL84was digested with restriction endonuclease HindIII, and the resultingHindIII overhangs were blunted using T4 DNA polymerase. The DNA samplewas then subjected to agarose gel electrophoresis to separate thefragments. The “vector” portion (carrying the origin of replication, thegene encoding spectinomycin resistance, and attP) was purified usingGeneClean (Bio 101), ligated using T4 DNA ligase, and transformed intoE. coli NovaBlue cells (Novagen). Plasmids isolated from three of theresulting transformants were tested by restriction endonuclease HindIIIor SalI digestion, and all three were confirmed to be pCL84 devoid ofthe Tc^(R) gene. One of these three plasmids was named pCL84(ΔTc, ΔH3)and was used in further procedures described below.

Construction of an ermC and tetR transcription fusion gene in pCL84(ΔTc,ΔH3)

[0056] The ermC gene, including its promoter, was amplified from plasmidpE194 (ATCC Number 68359) using Taq DNA polymerase with oligonucleotideprimers ermC-5′ (5′acgggtcgactcatatcttttattcaataatcg; SEQ ID NO: 1) andermC-3′ (5′ccggaaagcttacttattaaataatttatagc; SEQ ID NO: 2). The tetRgene was amplified from plasmid pWH354 (DE 3934454 Al; Geissendörfer, M.and W. Hillen, Appl. Microbiol. Biotechnol. 33:657-663, 1990) using TaqDNA polymerase with primers tetR-5′(5′taagtaagcttaaggaggaattaatgatgtctag; SEQ ID NO:3) and tetR-3′(5′acgggtcgacttaagacccactttcacatttaag; SEQ ID NO:4). The amplified tetRgene carried a synthetic ribosomal binding site. These two PCR productswere purified with the Wizard PCR Preparation Purification Kit(Promega), digested with HindIII restriction endonuclease, and ligatedtogether with T4 DNA ligase. The resulting ligated DNA was then used asthe template for PCR amplification using Taq DNA polymerase with primersermC-5′ and TetR-3′. The PCR product was purified with the Wizard PCRPreparation Purification Kit (Promega), digested with SalI restrictionendonuclease to make SalI ends, and then further purified with WizardPCR Preparation Purification Kit (Promega). The purified ermC-tetRfusion gene was then ligated to SalI-digested, shrimp alkalinephosphatase (SAP) treated, and gel purified pCL84(ΔTc, ΔH3). The ligatedDNA was transformed into E. coli NovaBlue cells (Novagen). Fifty-fivetransformants were subjected to colony PCR screening using primersermC-5′ and tetR-3′. Two clones, pCL84t #16, and #19, were identified ascontaining the ermC-tetR fusion gene. Plasmid DNA was purified fromthese two clones and the tetR gene sequence in clone #16 was confirmedby DNA sequencing. The ErmC gene provides not only a selectablephenotypic marker (erythromycin resistance), but also a constitutivepromoter for the TetR gene.

Integration of ermC-tetR Fusion Gene into the S. aureus Chromosome

[0057] Plasmid DNA isolated from clone #16 was transformed into S.aureus CYL316 cells by electroporation (Schenk, S. and R. A. Laddaga,FEMS Microbiology Letters 94:133-138, 1992). The transformation wasspread on LB plates containing 10 μg/ml of erythromycin. Sixtransformants were isolated and it was confirmed by a lipase assay (Lee,C. Y. et al, Gene 103:101-105, 1991) that ermC-tetR was integrated intothe phage L54a integration site on the chromosome in all six. Theresulting clones were designated strain CYL316t. The expected structurewas confirmed by PCR experiments.

Confirmation of tetR Expression in the CYL316t S. aureus Strain

[0058] Overnight cultures of E. coli DH5αPRO, and cultures of S. aureusRN4220 (Novick, R. P. et al., Methods in Enzymology 204:587-637, 1991)carrying pWH354, S. aureus strain CYL316t, or CYL316:pCL84 were diluted60-fold in 2 tubes of fresh LB containing 50 μg/ml spectinomycin (forDH5αPRO), 25 μg/ml kanamycin (for RN4220/pWH354), 10 μg/ml erythromycin(for CYL316t), or 3 μg/ml tetracycline (for CYL316:pCL84) and grown at37° C. At 2.5 hours, anhydrotetracycline was added to one set ofcultures to 200 ng/ml. The cells were grown for an additional 3 hours.The cells were pelleted from 3 ml of culture and resuspended in 200 82 Lof 1×PBS containing 50 μg/ml lysostaphin. After incubating at 4° C. for30 minutes, the cell suspensions were subjected to three freeze-thawcycles between a dry-ice/ethanol bath and a 37° C. water bath, followedby a 15-second sonication with a microtip. The cell lysates were thenboiled in SDS-PAGE sample buffer and subjected to electrophoresis on a12.5% SDS-polyacrylamide gel. The separated proteins were then blottedto a nitrocellulose membrane and analyzed by Western blot using ananti-Tet repressor protein antibody (kindly provided by Prof. WolfgangHillen). The results confirmed that tetR is constitutively expressed inS. aureus strain CYL316t.

Construction of a Staphylococcus aureus Strain that ConstitutivelyExpresses tetR as well as tetM

[0059] An EcoRI fragment containing the tet(M) gene of Tn916 wasisolated from plasmid pVB101, a construct similar to pVB201 (Burdett,V., J. Bacteriol. 175:7209-7265, 1993). The tet(M) gene fragment wascloned into the EcoRI site of pCL84t, resulting in plasmid pCL84tt.pCL84tt was integrated into the S. aureus chromosome in CYL316,resulting in strain CYL316tt. CYL316tt can grow in LB medium containing10 μg/ml tetracycline.

Example 2 Construction of Expression Plasmids The Steps in PlasmidConstruction are Diagramed in FIG. 2.

[0060] A. pWH353 and pWH354 Derivatives

[0061]E. coli/Bacillus subtilis shuttle expression vectors pWH353 andpWH354 were obtained from Professor Wolfgang Hillen (MikrobielleGenetik, Universität Tübingen, Tübingen, Germany; DE 3934454 Al;Geissendorfer, M. and W. Hillen, Appl. Microbiol. Biotechnol.33:657-663, 1990). These expression vectors carry a Tn10 tetR geneencoding Tet repressor. They also contain synthetic promoters with oneor two Tet repressor binding sites that are optimized for inducibleexpression in B. subtilis. These inducible promoters direct theexpression of a chloramphenicol acetyltransferase (CAT) gene in eachcase.

[0062] The CAT gene in pWH353 or pWH354 was replaced with a GST gene.The GST gene was PCR amplified with Taq DNA polymerase using pGEX-4T-2as the template and the combination of oligonucleotide primers 2a-5′-GST(5′ CTC GGT ACC GAG CTA AAA TTC GGA GGC ATA TCA AAT GAG CTC TGG) (SEQ IDNO:5), 2b-5′-GST (5′ GGC ATA TCA AAT GAG CTC TGG AGG TGG AGG CAT GTC CCCTAT AC) (SEQ ID NO:6), and 3′-GST-AvrII (5′ AGG CCT AGG TTA ATC CGA TTTTGG AGG ATG G) (SEQ ID NO:7) as the primers. The PCR fragment was clonedinto pT7Blue(T) vector (Novagen). The GST gene in the resultingpT7Blue(T) vector was then excised by KpnI and StuI double-endonucleasedigestion and further cloned into the KpnI/StuI sites of pWH353 andpWH354, resulting in pWH353-2G, and pWH354-2G. A synthetic gene encodingpeptide JT101 was obtained by annealling oligonucleotides Ml.Sac.a (5°CTG ATC CGA ATA CGT GGC AGT TGC GGT GGC CTA TGC ATA GCT) (SEQ ID NO:8)and M1. Sac.b (5′ ATG CAT AGG CCA CCG CAA CTG CCA CGT ATT CGG ATC AGAGCT) (SEQ ID NO:9) and cloned into the SacI site of pWH353-2G andpWH354-2G. Clones pWH353-2G-M1 and pWH354-2G-M1 were identified thatcontained the M1-GST fusion gene.

[0063] B. pWM353(ΔtetR) and pWH354(ΔtetR) and their Derivatives

[0064] Plasmids pWM353, pWH354 and pWH354-2G were each digested withrestriction endonucleases SpeI and BstEII, which excise a DNA fragmentencompassing the tetR gene in these two plasmids. The overhangs of theresulting DNA fragments were blunted using T4 DNA polymerase. The“vector” portion of each of these two digestions (the remainder of theplasmid without tetR) was gel purified, self-ligated using T4 DNAligase, and was then transformed into E. coli NovaBlue cells (Novagen).Clones carrying pWH353 or pWH354 or pWH354-2G deleted for the tetR genewere identified by PCR colony screening, and were designatedpWH353(ΔtetR), pWH354(ΔtetR) and pWH354-2G(ΔtetR), respectively.

[0065] C. pC³859 and its Derivatives

[0066] The following four oligonucleotides were chemically synthesized:

Tet-20F: 5′tcgagttcatgaaaaactaaaaaaaatattgacatccctatcagtgat (SEQ IDNO:10)

Tet2/3 OF: 5′agagataattaaaataatccctatcagtgatagagagcttgcatg (SEQ IDNO:11)

Tet2/3 OR: 5′caagctctctatcactgatagggattatt (SEQ ID NO:12)

Tet2OR: 5′ttaattatctctatcactgatagggatgtcaatattttttagtttttcatgaac (SEQ IDNO:13)

[0067] Oligonucleotides Tet-2OR and Tet-2/3OF were phosphorylated usingT4 polynucleotide kinase. Equal molar amounts of the phosphorylatedTet-2OR, Tet-2/3OF, and unphosphorylated Tet-2OF, Tet-2/3OR were mixedtogether, heated to 85° C. for 5 minutes and cooled down to roomtemperature gradually in a 2-hour period. The annealed oligonucleotides,called Tet-2O, comprised a synthetic promoter with two tet2O operatorsequences that are derived from Tn10.

[0068] pWH354(ΔtetR) was digested with restriction endonucleases XhoIand SphI, which excised the promoter/operator that controlled CATexpression. The digested DNA was separated on a 1% agarose gel. The DNAfragment of the vector portion was purified and ligated with Tet-20 DNAfragment. The ligation was transformed into DH5αPRO (Clontech). A cloneof the desired recombination was identified and designated pC³859.

[0069] The CAT gene open reading frame in pC³859 was substituted withthe GST gene amplified from plasmid pGEX-4T-2 (Pharmacia), greenfluorescence protein (GFP) gene amplified from pQBI63 (QuantumBiotechnologies), or maltose binding protein amplified from pMAL-c2 (NewEngland Biolabs), resulting in plasmid pC³859-GST, pC³859-GFP, andpC³859-MBP respectively.

[0070] D. pC³876 and pC³878

[0071] The GST gene was PCR amplified with Taq DNA polymerase fromplasmid pGEX-4T-2 with the following 3 primers: #1-5′-GST(TetR):5′aataaaaaactagtttgacaaataactctatca (SEQ ID NO:14)atgatagagtgtcacaaaaaggagg #2-5′-GST(TetR):5′gatagagtgtcaacaaaaaggaggaattaatga (SEQ ID NO:15)tgtcccctatactaggttattgg 3′-GST(TetR):5′ggattaaggtaaccttaatccgattttggagga (SEQ ID NO:16) tgg

[0072] #2-5′-GST(TetR) can anneal to the 5′ end of GST and amplify theGST gene along with the 3′-GST(TetR) primer; #1-5′-GST(TetR) can annealto the 5′ end of the resulting DNA fragment and extend the 5′ endsequence. The final PCR product contains the GST gene fused to thepromoter region used to control TetR expression in pWH353 or pWH354, andit has an SpeI site at its 5′ end and a BstEII site at its 3′ end. Theamplified DNA was purified with the Wizard PCR Preparation andPurification Kit (Promega) and cloned into the pT7Blue-3 vector(Novagen), resulting in a clone designated pT7Blue-3-GST. The cloned DNAfragment in pT7Blue-3-GST was then excised from the vector withrestriction endonucleases SpeI and BstEII, gel purified, and cloned intothe SpeI/BstEII sites of pWH353 and pWH354, resulting in plasmids pC³876and pC³878, respectively. These two plasmids have the same sequence aspWH353 or pWH354, except that the GST open reading frame sequence hasbeen substituted for the TetR open reading frame in the originalplasmids.

[0073] E. pC³874, pC³875, and pC³875 Derivatives

[0074] As the multilinker cloning site in pT7Blue-3 is flanked by twoEcoRI sites, the cloned GST DNA fragment in pT7Blue-3-GST was alsoexcised from pT7Blue-3-GST with EcoRI digestion. The agarosegel-purified DNA fragment was then ligated to the EcoRI vector portionof pC³859 containing the origin of replication. The resulting plasmidspC³874 and pC³875 have the GST gene in alternative orientations.

[0075] The following DNA sequence, which encoded the JT01 peptide(DPNTWQLRWPMH; SEQ ID NO:17) followed by a Gly-Gly-Arg-Gly-Gly-Met (SEQID NO: 18) linker, was inserted after the initiation ATG codon of theGST gene in pC³875, resulting in plasmid pC³882:

5′gatcctaatacatggcagttgaggtggcctatgcatggcggccgcggaggtatg (SEQ ID NO:19).

[0076] The following DNA sequence, which encoded JT01 peptide flanked bya Ser-Ser dipeptide on each side (ssJT01ss) and followed by aGly-Gly-Arg-Gly-Gly-Met linker, was inserted after the initiation ATGcodon of the GST gene in pC³875, resulting in plasmid pC³883:

5′agctctgatcctaatacatggcagttgaggtggcctatgcattcttcaggcggccgcggaggtatg(SEQ ID NO:20)

[0077] The following DNA sequence, which encoded a peptide with thesequence ofSer-Arg-Trp-Glu-Lys-Tyr-Ile-Asn-Ser-Phe-Glu-Leu-Asp-Ser-Arg-Gly-Gly-Arg-Gly-Gly-Met(LysN2), was inserted after the initiation ATG codon of the GST gene inpC³875, resulting in plasmid pC³888:

5′tctagatgggaaaaatatattaattcttttgaattagattctcgaggtggtagaggtggaatg (SEQID NO:21)

[0078] The following DNA sequence, which encoded a peptide with thesequence ofSer-Ser-Gln-Gly-Thr-Met-Arg-Trp-Phe-Asp-Trp-Tyr-Arg-Ser-Arg-Gly-Gly-Arg-Gly-Gly-Met(LysN3), was inserted after the initiation ATG codon of the GST gene inpC³875, resulting in plasmid pC³889:

5′agctctcaaggtactatgagatggtttgattggtatagatctcgaggtggtagaggtggaatg (SEQID NO:22)

[0079] F. pC³884 and pC³886

[0080] The green fluorescence protein (GFP) gene was amplified fromplasmid pQBI63 (Quantum Biotechnologies Inc.) using the followingoligonucleotides with Taq DNA polymerase:

5GFP(NotI): 5′agcaccttggcggccgcggaggtgctagcaaaggagaagaactcttcac (SEQ IDNO:23)

3′GFP(BstEII): 5′aactgaggtaacctcagttgtacagttcatccatgcc (SEQ ID NO:24).

[0081] The amplified DNA was purified with the Wizard PCR Preparationand Purification Kit, and digested with restriction endonucleases NotIand BstEII. The digested DNA was gel purified and ligated to theNotlI/BstEII sites of pC³882, resulting in pC³886.

[0082] The maltose binding protein (MBP) gene was amplified from plasmidpMAL-c2 (New England Biolabs) with Taq DNA polymerase using thefollowing oligonucleotides:

5′MalE(NotI): 5′tttaccttggcggccgcggaggtaaactgaagaaggtaaactggtaatctgg(SEQ ID NO:25);

3′MalE(BstEII): 5′acttagggtaaccttaagtctgc gcgtctttcagggcttc (SEQ IDNO:26)

[0083] The amplified DNA was purified with the Wizard PCR Preparationand Purification Kit, and digested with restriction endonucleases NotIand BstEII. The digested DNA was gel purified and ligated to theNotI/BstEII sites of pC³882, resulting in pC³884.

Example 3 Characterization of Expression Plasmids

[0084] A. pWH353 and pWH354

[0085] pWH353 and pWH354 were transformed separately into S. aureusRN4220 cells by electroporation. The transformants were tested forinducible expression by growing in LB broth containing 30 μg/mlkanamycin. After the OD₆₀₀ reached 0.5, the cultures were split andtetracycline was added to one set of the cultures to a finalconcentration of 0.5 μg/ml. The induction was for 3 hours at 37° C. TheS. aureus cells were then pelleted and resuspended in 80 mM Tris-HCl, pH7.4 containing 200 μg/ml lysostaphin, incubated at 37° C. for 5 minutes,frozen and thawed twice in a dry ice/ethanol bath and in a 37° C. waterbath. The samples were then sonicated twice and centrifuged at 14,000 gfor 10 minutes, and the supernatants were collected and subjected toanalysis on a 4-20% acrylamide gradient gel, by SDS-PAGE. The CATactivities in these 25 samples were determined and summarized inTable 1. The results indicated that CAT is inducibly expressed, althoughthe expression levels are not high enough to produce a visible band byCoomassie staining on an SDS polyacrylamide gel. TABLE 1 Inducibleexpression of chloramphenicol acetyltransferase activity in S. aureusPlasmid Induction Relative CAT Activity No Plasmid − ND No Plasmid + NDpWH353 − 41 pWH353 + >4110 pWH354 − 1 pWH354 + 350

[0086] Expression of GST with pWH353-2G in S. aureus RN4220 was alsocharacterized. The transformants of pWH353-2G were tested for inducibleexpression by growing in LB broth containing 30 μg/ml kanamycin. AfterOD₆₀₀ reached 0.5, the cultures were divided into 4 tubes andanhydrotetracycline was added to 0, 0.0625, 0.25, or 1 μg/ml. Theinduction was for 3 hours at 37° C. The S. aureus cells were thenpelleted and resuspended in 1×PBS containing 100 μg/ml lysostaphin,incubated at 37° C. for 5 minutes, frozen and thawed twice on dryice/ethanol and 37° C. water bath. The samples were then sonicated twiceand centrifuged at 14,000 g for 10 minutes and the supernatants werecollected and subjected to analysis on a 4-20% SDS-PAGE. The resultindicated that GST expression level was very low, not detectable byCoomassie blue staining. Characterization of pWH354-2G, pWH353-2G-Ml,and pWH354-2G-M1 transformants of RN4220 generated similar results.

[0087] B. Expression and Growth characterization of S. aureus CellsHarboring pWH353(ΔtetR) and pWH354(ΔtetR), or pWH354-2G(ΔtetR)

[0088] pWH353(ΔtetR), pWH354(ΔtetR) and pWH354-2G(ΔtetR) weretransformed into separate cultures of S. aureus CYL316t cells byelectroporation. Overnight cultures of CYL316t/pWH353(ΔtetR),CYL316t/pWH354(ΔtetR) and CYL316t/pWH354-2G(ΔtetR) were diluted 100-foldin fresh LB with 5 μg/ml erythromycin and 25 μg/ml kanamycin. After theOD₆₀₀ reached 0.5, the cultures were split into two tubes, andanhydrotetracycline was added to one set of the cultures to a finalconcentration of 0.2 μg/ml. The induction was for 3 hours at 37° C. TheS. aureus cells were then pelleted and resuspended in 1×PBS containing100 μg/ml lysostaphin, incubated at 37° C. for 5 minutes, frozen andthawed twice in a dry ice/ethanol bath and in a 37° C. water bath. Thesamples were then sonicated twice and centrifuged at 14,000 g for 10minutes. The supernatants were collected and subjected to analysis on a4-20% acrylamide gradient gel, by SDS-PAGE. Cultures ofCYL316t/pWH353(ΔtetR) were also tested, with similar results. Theresults indicated that removing the tetR gene from the pWH353 and pWH35⁴plasmids drastically increased efficiency of CAT expression, to thepoint where a protein band became visible by Coomassie blue staining onan SDS-polyacrylamide gel, while GST expression was not significantlyenhanced.

[0089] Growth of CYL316t, CYL316t/pWH353(ΔtetR), CYL316t/pWH354(ΔtetR)and CYL316t/pWH354-2G(ΔtetR) was also characterized by the followingexperiment. Overnight cultures of CYL316t, CYL316t/pWH353(ΔtetR),CYL316t/pWH354(ΔtetR) and CYL316t/pWH354-2G(ΔtetR) were diluted 100-foldin fresh tryptone-soy broth (TSB; Difco) containing 5 μg/ml erythromycinand 25 μg/ml kanamycin (for CYL316t/pWH353(ΔtetR), CYL316t/pWH354(ΔtetR)and CYL316t/pWH354-2G(ΔtetR)). After growing for 1 hour at 37° C, eachculture was split into two tubes and to one anhydrotetracycline wasadded to 200 ng/ml. The OD₆₀₀'s of the cultures were then recorded atvarious time points. The results depicted in FIGS. 3A-3D demonstratethat anhydrotetracycline induction caused a significant inhibition in S.aureus growth.

[0090] C. Growth Characterization of S. aureus Cells Carrying pC³859

[0091] An overnight culture of CYL316t/pC³859 was diluted 100-fold infresh TSB with 5 μg/ml erythromycin and 25 μg/ml kanamycin. Aftergrowing for 1 hour at 37° C., the culture was split into two tubes. Toone, anhydrotetracycline was added to 200 ng/ml. The OD₆₀₀ of theculture was then recorded at various time points. The result is depictedin FIG. 3E, which demonstrates that the anhydrotetracycline inductionrelated cytotoxicity observed with S. aureus cells carryingpWH353(ΔtetR) or pWH354(ΔtetR) is much reduced for S. aureus cellsharboring pC³859, by comparison.

[0092] D. Expression Characterization of E. coli or S. aureus cellsharboring pC³859 or its Derivatives

[0093] Plasmids pC³859, pC³859-GST, pC³859-GFP, and pC³859-MBP were eachtransformed into separate cultures of E. coli DH5αPRO and S. aureusCYL316t. Overnight cultures of the resulting E. coli or S. aureustransformant cells were diluted 100-fold in fresh LB with 50 μg/mlspectinomycin and 25 μg/ml kanamycin for E. coli cells or LB with 5μg/ml erythromycin and 25 μg/ml kanamycin for S. aureus cells. After theOD₆₀₀ reached 0.5, the cultures were split into two tubes, andanhydrotetracycline was added to one set of the cultures to 0.2 μg/ml.The induction was for 3 hours at 37° C. The E. coli cells were pelletedand boiled 5 minutes in SDS-PAGE sample buffer. The S. aureus cells werepelleted and resuspended in 1 ×PBS containing 100 μg/ml lysostaphin,incubated at 37° C. for 5 minutes, frozen and thawed twice in a dryice/ethanol bath and in a 37° C. water bath. The samples were thensonicated twice and centrifuged at 14,000 g for 10 minutes and thesupernatants were mixed with SDS-PAGE sample buffer and boiled for 5minutes. The samples were subjected to analysis on a 4-20% acrylamidegradient gel, by SDS-PAGE. The results indicated that while CAT can beefficiently expressed from pC³859 in S. aureus, other genes, such asGST, GFP, or MBP are not efficiently expressed using the same geneticbackground.

[0094] E. Expression Characterization of S. aureus Cells HarboringpC³876, pC³878, pC³874, or pC³875

[0095] Plasmids pC³876, pC³878, pC³874, and pC³875 were transformed intoseparate cultures of S. aureus CYL316t. Overnight cultures of theresulting S. aureus cells were diluted 100-fold in fresh LB with 5 μg/mlerythromycin and 25 μg/ml kanamycin for S. aureus cells. After the OD₆₀₀reached 0.5, the cultures were split into two tubes andanhydrotetracycline was added to one set of the cultures to 0.2 μg/ml.The induction was for 3 hours at 37° C. The S. aureus cells werepelleted and resuspended in 80 mM Tris-HCl, pH 7.4 containing 200 μg/mllysostaphin, incubated at 37° C. for 5 minutes, frozen and thawed twicein a dry ice/ethanol bath and in a 37° C. water bath. The samples werethen sonicated twice and centrifuged at 14,000 g for 10 minutes and thesupernatants were mixed with SDS-PAGE sample buffer and boiled for 5minutes. The samples were subjected to analysis on a 4-20% acrylamidegradient gel, by SDS-PAGE. The results indicated that GST gene can beefficiently expressed with these constructs. GST from the above celllysates was also purified with glutathione-Sepharose, and analyzed bySDS-PAGE. The results indicated that pC³875 had the lowest level ofbasal expression from the tet promoter/operator.

[0096] F. Expression Characterization of pC³882 and pC³883 in CYL316t

[0097] Plasmid pC³882 and pC³883 were transformed into separate culturesof S. aureus CYL316t. Overnight cultures of the resulting S. aureustransformant cells were diluted 100-fold in fresh LB with 5 μg/mlerythromycin and 25 μg/ml kanamycin. After the OD₆₀₀ reached 0.5, thecultures were split into two tubes and anhydrotetracycline was added toone set of the cultures to 0.2 μg/ml. The induction was for 3 hours at37° C. The S. aureus cells were pelleted and resuspended in 1×PBScontaining 100 μg/ml lysostaphin, incubated at 37° C. for 5 minutes, andfrozen and thawed twice in a dry ice/ethanol bath and in a 37° C. waterbath. The samples were then sonicated twice and centrifuged at 14,000 gfor 10 minutes, and the supernatants were mixed with SDS-PAGE samplebuffer and boiled for 5 minutes. The samples were subjected to analysison a 4-20% acrylamide gradient gel, by SDS-PAGE. The results indicatedthat both JT01-GST and ssJT01ss could be efficiently expressed, and theexpression for JT01-GST was significantly higher than ssJT01ss-GST. Withan ELISA assay (Pharmacia; product #27-4592), it was determined thatexpression of JT01-GST increased about 1000-fold uponanhydrotetracycline induction.

[0098] JT01-GST and ssJT01ss-GST fusion polypeptides were also purifiedfrom the above described cell extracts using glutathione affinity resin.The purified peptide-GST fusion polypeptides were tested for theirinhibitory activity on S. aureus MetRS with 2 mM ATP, 50 μM methionine,and 90 μM E. coli total tRNA as the substrates. Under these conditions,JT101-GST only inhibited 60% of S. aureus activity with an IC₅₀ (peptideconcentration required to inhibit 50% MetRS activity) about 15 nM, whileboth free JT101 peptide and ssJT01 ss-GST could fully inhibit S. aureusMetRS activity with an IC₅₀ of about 100 nM.

[0099] G. Growth Characterization of S. aureus Cells Carrying PlasmidpC³882 or pC³883

[0100] Plasmids pC³882 and pC³883 were transformed separately into S.aureus strains CYL316t and CYL316tt. Overnight cultures of the resultingS. aureus transformant cells grown in TSB with 5 μg/ml erythromycin and25 μg/ml kanamycin were diluted 100-fold in fresh TSB with 5 μg/mlerythromycin and 25 μg/ml kanamycin. After growing for 1 hour at 37° C.,the culture was split into two tubes and to one, anhydrotetracycline(for CYL316t strains) or tetracycline (for CYL316tt strains) was addedto 200 ng/ml and 1 μg/ml, respectively. The OD₆₀₀ of the culture wasthen recorded at various time points. The results depicted in FIGS. 5A,5B, and 5C demonstrate that induction of expression of JT01-GST orssJT01ss-GST, but not GST alone, caused significant inhibition of S.aureus growth. Between the two fusion polypeptides, ssJT01ss-GSTexpression had the stronger growth inhibitory effect.

[0101] H. Expression of JT01-GFP, JT01-MBP Fusions

[0102] Plasmid pC³884 and pC³886 were each transformed separately intoseparate cultures of S. aureus CYL316tt. Overnight cultures of theresulting transformant S. aureus cells were diluted 100-fold in fresh LBwith 5 μg/ml erythromycin and 25 μg/ml kanamycin. After the OD₆₀₀reached 0.5, the cultures were split into two tubes and tetracycline wasadded to one set of the cultures to 1 μg/ml. The induction was for 3hours at 37° C. The S. aureus cells were pelleted and resuspended in1×PBS containing 100 μg/ml lysostaphin, incubated at 37° C. for 5minutes, frozen and thawed twice in a dry ice/ethanol bath and in a 37°C. water bath. The samples were then sonicated twice and centrifuged at14,000 g for 10 minutes and the supernatants were mixed with SDS-PAGEsample buffer and boiled for 5 minutes. Analysis of the resultingsamples on a SDS-polyacrylamide gel revealed that both JT01-GFP andJT01-MBP polypeptides were efficiently expressed.

[0103] I. Expression of LysN2-GST, and LysN3-GST Fusions

[0104] Plasmid pC³888 or pC³889 was transformed into S. aureus CYL316t.Overnight cultures of the resulting S. aureus cells were diluted100-fold in fresh LB with 5 μg/ml erythromycin and 25 μg/ml kanamycin.After the OD₆₀₀ reached 0.5, the cultures were split into two tubes andanhydrotetracycline was added to one set of the cultures to 0.2 μg/ml.The induction was for 3 hours at 37° C. The S. aureus cells werepelleted and resuspended in 1×PBS containing 100 μg/ml lysostaphin,incubated at 37° C. for 5 minutes, frozen and thawed twice in a dryice/ethanol bath and in a 37° C. water bath. The samples were thensonicated twice and centrifuged at 14,000 g for 10 minutes, and thesupernatants were mixed with SDS-PAGE sample buffer and boiled for 5minutes. Examination of the resulting samples on a SDS-polyacrylamidegel revealed that both LysN2-GST and LysN3-GST were efficientlyexpressed.

Example 4 Protection of a Lethal S. aureus Infection by IntracellularExpression of a Peptide In Vivo

[0105] In experiments using S. aureus cells grown in culture,intracellularly expressed ssJT01ss bound to and inhibited a specificessential cellular target in a manner similar to that of anantimicrobial drug. Hence induction of expression of this peptide duringan infection should have the effect of an antibiotic. An establishedanimal infection model was used to test this concept (Onyegji, C. O. etal., Antimicrobial Agents and Chemotherapy 38:112-117, 1994).

[0106] Six groups of CD-1 female mice (5 mice per group, Charles RiverLaboratories, Wilmington, Mass.) weighing 20-24 grams were used in thisexperiment. The inoculum was prepared from CYL316tt/pC³883 (encoding assJT01ss peptide-GST fusion protein under the control of the tet operon)which was cultured at 37° C. for 17 hours in TS broth containingerythromycin and kanamycin. 1.6×10¹⁰ cfu (colony-forming units) of S.aureus CYL316tt/pC³883 (OD₆₀₀ of0.1=10⁸ cfu/ml) from the overnightculture were diluted to 20 ml with 0.01 M PBS (Sigma P-0261) containing8% hog gastric mucin (Sigma M-2378) as well as 50 μg/ml kanamycin and 10μg/ml erythromycin. Each mouse of groups 1 through 4 was injected with0.5 ml of the inoculum intraperitoneally (i.p.), equivalent to 4×10⁸cfu/mouse (lethal dose). Groups 5 and 6 served as vector controls. Eachmouse of these two groups was injected with 4×10⁸ cfu of CYL316tt/pC³875, which was cultured and processed the same way asCYL316tt/pC³883. One half hour and four hours after the inoculation,groups 1 and 5 received a saline injection i.p. at 10 ml/kg; groups 2and 6 received i.p. injections of tetracycline (Sigma T-3383) at 8mg/kg; group 3 received i.p. injections of tetracycline at 4 mg/kg;group 4 received i.p. injections of ciprofloxacin (Bayer 851510,dissolved in water) at 50 mg/kg. The injection volume for all theanimals was 10 ml/kg. Surviving mice were counted at 7 days postinoculation. Ciprofloxacin given at 50 mg/kg protected all infectedanimals from lethal infection.

[0107] The data summarized in Table 2 demonstrate that induction ofintracellular expression of the ssJT01ss peptide can be achieved in ananimal infection. Inhibition of S. aureus MetRS by the intracellularlyexpressed ssJT01ss peptide cured a lethal infection in a mouse model.TABLE 2 Inhibition of S. aureus growth in mice by intracellularproduction of S. aureus MetRS inhibitor # of Mice # of Mice ExperimentalCondition Tested Survival M1 Peptide Saline, 10 mg/kg C2 5 0 Inducer,mg/kg C2 5 5 Expression Control Saline, 10 mg/kg C2 5 0 Inducer, mg/kgC2 5 0

[0108] The relevant teachings of all references cited herein are herebyincorporated by reference herein in their entirety.

[0109] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1 28 1 33 DNA Artificial Sequence Oligonucleotide 1 acgggtcgactcatatcttt tattcaataa tcg 33 2 32 DNA Artificial SequenceOligonucleotide 2 ccggaaagct tacttattaa ataatttata gc 32 3 34 DNAArtificial Sequence Oligonucleotide 3 taagtaagct taaggaggaa ttaatgatgtctag 34 4 34 DNA Artificial Sequence Oligonucleotide 4 acgggtcgacttaagaccca ctttcacatt taag 34 5 45 DNA Artificial SequenceOligonucleotide 5 ctcggtaccg agctaaaatt cggaggcata tcaaatgagc tctgg 45 644 DNA Artificial Sequence Oligonucleotide 6 ggcatatcaa atgagctctggaggtggagg catgtcccct atac 44 7 31 DNA Artificial SequenceOligonucleotide 7 aggcctaggt taatccgatt ttggaggatg g 31 8 42 DNAArtificial Sequence Oligonucleotide 8 ctgatccgaa tacgtggcag ttgcggtggcctatgcatag ct 42 9 42 DNA Artificial Sequence Oligonucleotide 9atgcataggc caccgcaact gccacgtatt cggatcagag ct 42 10 48 DNA ArtificialSequence Oligonucleotide 10 tcgagttcat gaaaaactaa aaaaaatatt gacatccctatcagtgat 48 11 45 DNA Artificial Sequence Oligonucleotide 11 agagataattaaaataatcc ctatcagtga tagagagctt gcatg 45 12 29 DNA Artificial SequenceOligonucleotide 12 caagctctct atcactgata gggattatt 29 13 56 DNAArtificial Sequence Oligonucleotide 13 ttaattatct ctatcactga tagggatgtcaatatttttt ttagtttttc atgaac 56 14 58 DNA Artificial SequenceOligonucleotide 14 aataaaaaac tagtttgaca aataactcta tcaatgatagagtgtcacaa aaaggagg 58 15 56 DNA Artificial Sequence Oligonucleotide 15gatagagtgt caacaaaaag gaggaattaa tgatgtcccc tatactaggt tattgg 56 16 36DNA Artificial Sequence Oligonucleotide 16 ggattaaggt aaccttaatccgattttgga ggatgg 36 17 12 PRT Artificial Sequence Synthetic Peptide 17Asp Pro Asn Thr Trp Gln Leu Arg Trp Pro Met His 1 5 10 18 6 PRTArtificial Sequence Synthetic Peptide 18 Gly Gly Arg Gly Gly Met 1 5 1954 DNA Artificial Sequence Oligonucleotide 19 gatcctaata catggcagttgaggtggcct atgcatggcg gccgcggagg tatg 54 20 66 DNA Artificial SequenceOligonucleotide 20 agctctgatc ctaatacatg gcagttgagg tggcctatgcattcttcagg cggccgcgga 60 ggtatg 66 21 21 PRT Artificial Sequence peptide21 Ser Arg Trp Glu Lys Tyr Ile Asn Ser Phe Glu Leu Asp Ser Arg Gly 1 510 15 Gly Arg Gly Gly Met 20 22 63 DNA Artificial SequenceOligonucleotide 22 tctagatggg aaaaatatat taattctttt gaattagattctcgaggtgg tagaggtgga 60 atg 63 23 21 PRT Artificial Sequence peptide 23Ser Ser Gln Gly Thr Met Arg Trp Phe Asp Trp Tyr Arg Ser Arg Gly 1 5 1015 Gly Arg Gly Gly Met 20 24 63 DNA Artificial Sequence Oligonucleotide24 agctctcaag gtactatgag atggtttgat tggtatagat ctcgaggtgg tagaggtgga 60atg 63 25 49 DNA Artificial Sequence Oligonucleotide 25 agcaccttggcggccgcgga ggtgctagca aaggagaaga actcttcac 49 26 37 DNA ArtificialSequence Oligonucleotide 26 aactgaggta acctcagttg tacagttcat ccatgcc 3727 52 DNA Artificial Sequence Oligonucleotide 27 tttaccttgg cggccgcggaggtaaactga agaaggtaaa ctggtaatct gg 52 28 40 DNA Artificial SequenceOligonucleotide 28 acttagggta accttaagtc tgcgcgtctt tcagggcttc 40

What is claimed is:
 1. Gram-positive bacteria containing one or more tetrepressor genes and one or more genes conferring tetracyclineresistance.
 2. Bacteria of claim 1 which are staphylococci. 3.Gram-positive bacteria with a genotype including tetR and tet(M). 4.Gram-positive bacteria with a chromosomal genotype including tetR andtet(M).
 5. Staphylococcus aureus with a genotype including tetR and oneor more genes conferring tetracycline resistance.
 6. Staphylococcusaureus strain CYL316t.
 7. Staphylococcus aureus strain CYL316tt.